Intratumorally administered lactoferrin in the treatment of malignant neoplasms and other hyperproliferative diseases

ABSTRACT

The present invention relates to methods of treating a hyperproliferative disease by administering a composition of lactoferrin alone or in combination with standard anti-cancer therapies.

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/379,442 filed on May 10, 2002; U.S. Provisional Application No.60/379,441 filed on May 10, 2002 and U.S. Provisional Application No.60/379,474 filed on May 10, 2002, which are incorporated herein byreference in their entirety.

FIELD OF INVENTION

[0002] The present invention relates to methods of treating ahyperproliferative disease by administering a composition of lactoferrinalone or in combination with standard anti-cancer therapies. Thelactoferrin composition may be administered orally, intravenously,intratumorally, or topically.

BACKGROUND OF THE INVENTION

[0003] Currently, there are few effective options for the treatment ofmany common cancer types. The course of treatment for a given individualdepends on the diagnosis, the stage to which the disease has developed,and factors such as age, sex, and general health of the patient. Themost conventional options of cancer treatment are surgery, radiationtherapy, and chemotherapy. Surgery plays a central role in the diagnosisand treatment of cancer. Typically, a surgical approach is required forbiopsy and the removal of cancerous growth. However, if the cancer hasmetastasized and is widespread, surgery is unlikely to result in a cure,and an alternate approach must be taken. Side effects of surgery includediminished structural or organ function and increased risk of infection,bleeding, or coagulation related complications. Radiation therapy,chemotherapy, biotherapy and immunotherapy are alternatives to surgicaltreatment of cancer (Mayer, 1998; Ohara, 1998; Ho et al., 1998). Thedisadvantage of many of the alternative therapies are the side effects,which can include myelosuppression, skin irritation, difficultyswallowing, dry mouth, nausea, diarrhea, hair loss, weight loss, andloss of energy (Curran, 1998; Brizel, 1998).

[0004] Lactoferrin is a single chain metal binding glycoprotein. Manycells types, such as monocytes, macrophages, lymphocytes, and intestinalbrush-border cells, are known to have lactoferrin receptors. In additionto lactoferrin being an essential growth factor for both B and Tlymphocytes, lactoferrin has a wide array of functions related to hostprimary defense mechanisms. For example, lactoferrin has been reportedto activate natural killer (NK) cells, induce colony stimulatingactivity, activate polymorphonuclear neutrophils (PMN), regulategranulopoeisis, enhance antibody-dependent cell cytotoxicity, stimulatelymphokine-activated killer (LAK) cell activity, and potentiatemacrophage toxicity.

[0005] Recently, bovine lactoferrin (bLF) was used as a prophylaxis fortumor formation and/or established tumors. The present invention is thefirst to use lactoferrin as a treatment, not a prophylaxis, forestablished tumors.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention is directed to a method for treating ahyperproliferative disease. The method of treatment involvesintratumoral administration of lactoferrin.

[0007] An embodiment of the present invention is a method of treating ahyperproliferative disease comprising the step of administeringintratumorally to a subject a lactoferrin composition in an amountsufficient to provide an improvement in the hyperproliferative disease.The amount of the lactoferrin composition that is administered is about0.1 μg to about 10 μg per day.

[0008] The lactoferrin composition is dispersed in a pharmaceuticallyacceptable carrier. More particularly, the lactoferrin is mammalianlactoferrin, for example, human or bovine. In specific embodiments, thelactoferrin is recombinant lactoferrin.

[0009] The hyperproliferative disease is further defined as cancer,which comprises a neoplasm. The neoplasm is selected from the groupconsisting of melanoma, non-small cell lung, small-cell lung, lunghepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma, leukemia,neuroblastoma, squamous cell, head, neck, gum, tongue, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,sarcoma, cervical, gastrointestinal, lymphoma, brain, colon, andbladder.

[0010] Yet further, the hyperproliferative disease is selected from thegroup consisting of rheumatoid arthritis, inflammatory bowel disease,osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas,vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions,carcinoma in situ, oral hairy leukoplakia, and psoriasis.

[0011] Another embodiment is a method of treating a hyperproliferativedisease comprising the step of supplementing a systemic and/or localimmune system in a subject by increasing the amount of lactoferrin inthe vicinity of the hyperproliferative disease. The lactoferrin isadministered intratumorally. The lactoferrin enhances, stimulates'and/or up-regulates interleukin-18 and Granulocyte Macrophage ColonyStimulating Factor (GM-CSF). It is envisioned that interleukin-18stimulates the production or activity of immune cells, for example Tlymphocytes or natural killer cells and GM-CSF promotes the migrationand maturation of immune cells including dendritic and other antigenpresenting cells.

[0012] Another embodiment is a method of enhancing a local immuneresponse in the vicinity of a tumor following the step of administeringintratumorally to the subject a lactoferrin composition. It isenvisioned that the lactoferrin composition stimulates interleukin-18and/or GM-CSF in the site of injection, which stimulates the productionor activity of immune cells, e.g., T lymphocytes or natural killercells. T lymphocytes are selected from the group consisting of CD4+,CD8+ and CD3+ cells.

[0013] In specific embodiments, the present invention is drawn to amethod of treating a hyperproliferative disease comprising administeringintratumorally to a subject a lactoferrin composition in combinationwith chemotherapy, biotherapy, immunotherapy, surgery or radiotherapy.

[0014] The foregoing has outlined rather broadly the features andtechnical advantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a more complete understanding of the present invention,reference is now made to the following descriptions taken in conjunctionwith the accompanying drawings.

[0016]FIG. 1 shows squamous cell tumor growth with and without oral,intravenous and intratumoral administration of recombinant humanlactoferrin.

[0017]FIG. 2 shows percent tumor growth inhibition in animals receivinglactoferrin, cisplatin and lactoferrin in combination with cisplatin.

[0018]FIG. 3 shows the percent tumor growth inhibition with lactoferrinin combination with various doses of cisplatin.

[0019]FIG. 4 shows the NK activity after treatment with lactoferrin.

[0020]FIG. 5 shows squamous cell tumor growth with and withoutintratumoral administration of recombinant lactoferrin once or twice aday.

DETAILED DESCRIPTION OF THE INVENTION

[0021] It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

[0022] As used herein, the use of the word “a” or “an” when used inconjunction with the term “comprising” in the claims and/or thespecification may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

[0023] The term “hyperproliferative disease” as used herein refers toany disease or disorder in which the cells proliferate more rapidly thannormal tissue growth. Thus, a hyperproliferating cell is a cell that isproliferating more rapidly than normal cells.

[0024] The term “parenteral administration” as used herein includes anyform of administration in which the compound is absorbed into thesubject without involving absorption via the intestines. Exemplaryparenteral administrations that are used in the present inventioninclude, but are not limited to intramuscular, intravenous,intraperitoneal, intratumoral, intraocular, or intraarticularadministration.

[0025] The term “intravenous administration” as used herein includes alltechniques to deliver a lactoferrin composition to the systemiccirculation via an intravenous injection or infusion.

[0026] The term “intratumoral administration” as used herein includesall techniques to deliver a lactoferrin composition to the site of atumor including injection, electroporation, creams, lotions or otherforms of administration.

[0027] The term “oral administration” as used herein includes oral,buccal, enteral or intragastric administration.

[0028] The term “topical administration” as used herein includesapplication to a dermal, epidermal, subcutaneous or mucosal surface.

[0029] The term “pharmaceutically acceptable carrier” as used hereinincludes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well know in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

[0030] The term “lactoferrin” or “LF” as used herein refers to native orrecombinant lactoferrin. Native lactoferrin can be obtained bypurification from mammalian milk or colostrum or from other naturalsources. Recombinant lactoferrin (rLF) can be made by recombinantexpression or direct production in genetically altered animals, plants,fungi, bacteria, or other prokaryotic or eukaryotic species, or throughchemical synthesis.

[0031] The term “subject” as used herein, is taken to mean any mammaliansubject to which the lactoferrin composition is administered accordingto the methods described herein. In a specific embodiment, the methodsof the present invention are employed to treat a human subject. Anotherembodiment includes treating a human subject suffering from ahyperproliferative disease.

[0032] The term “therapeutically effective amount” as used herein refersto an amount that results in an improvement or remediation of thesymptoms of the disease or condition.

[0033] The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of alactoferrin composition so that the subject has an improvement in thedisease. The improvement is any improvement or remediation of thesymptoms. The improvement is an observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thedisease condition, but may not be a complete cure for the disease.Specifically, improvements in patients with cancer may include tumorstabilization, tumor shrinkage, increased time to progression, increasedsurvival or improvements in the quality of life. Beneficial effect mayalso be reflected in an improvement of the patient's immune system asmeasured by the number and activity of circulating immune cells such asCD4+ cells, CD8+ cells, NK cells and CD40+ cells.

[0034] The term “vicinity” as used herein refers to in or around thearea or site of the tumor and/or hyperproliferative disease. Forexample, “vicinity of a tumor” may refer to the area in or around thetumor or margins of the tumor. Vicinity includes the area adjacent tothe tumor, the area over the tumor, the area under the tumor, the marginarea around the tumor, or the area adjacent the tumor margin area.

A. Pharmaceutical Compositions

[0035] The lactoferrin used according to the present invention can beobtained through isolation and purification from natural sources, forexample, but not limited to mammalian milk. The lactoferrin ispreferably mammalian lactoferrin, such as bovine or human lactoferrin.In preferred embodiments, the lactoferrin is human lactoferrin producedrecombinantly using genetic engineering techniques well known and usedin the art, such as recombinant expression or direct production ingenetically altered animals, plants or eukaryotes, or chemicalsynthesis. See, i.e., U.S. Pat. Nos. 5,571,896; 5,571,697 and 5,571,691,which are herein incorporated by reference.

[0036] Administration of the lactoferrin compositions according to thepresent invention will be via any common route, orally, parenterally, ortopically. Exemplary routes include, but are not limited to oral, nasal,buccal, rectal, vaginal, intramuscular, intraperitoneal, intravenous,intraarterial, intratumoral or dermal. Such compositions would normallybe administered as pharmaceutically acceptable compositions as describedherein.

[0037] The compositions of the present invention may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

[0038] Sterile injectable solutions are prepared by incorporating thelactoferrin in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0039] Further in accordance with the present invention, the inventivecomposition suitable for oral administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable or edible and includes liquid,semi-solid, i.e., pastes, or solid carriers. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of a lactoferrinpreparation contained therein, its use in an orally administrablelactoferrin for use in practicing the methods of the present inventionis appropriate. Examples of carriers or diluents include fats, oils,water, saline solutions, lipids, liposomes, resins, binders, fillers andthe like, or combinations thereof.

[0040] In accordance with the present invention, the composition iscombined with the carrier in any convenient and practical manner, i.e.,by solution, suspension, emulsification, admixture, encapsulation,microencapsulation, absorption and the like. Such procedures are routinefor those skilled in the art.

[0041] In a specific embodiment of the present invention, thecomposition in powder form is combined or mixed thoroughly with asemi-solid or solid carrier. The mixing can be carried out in anyconvenient manner such as grinding. Stabilizing agents can be also addedin the mixing process in order to protect the composition from loss oftherapeutic activity through, i.e., denaturation in the stomach.Examples of stabilizers for use in an orally administrable compositioninclude buffers, antagonists to the secretion of stomach acids, aminoacids such as glycine and lysine, carbohydrates such as dextrose,mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,mannitol, etc., proteolytic enzyme inhibitors, and the like. Morepreferably, for an orally administered composition, the stabilizer canalso include antagonists to the secretion of stomach acids.

[0042] Further, the composition for oral administration which iscombined with a semi-solid or solid carrier can be further formulatedinto hard or soft shell gelatin capsules, tablets, or pills. Morepreferably, gelatin capsules, tablets, or pills are enterically coated.Enteric coatings prevent denaturation of the composition in the stomachor upper bowel where the pH is acidic. See, i.e., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, i.e., epithelial enterocytes and Peyer'spatch M cells.

[0043] In another embodiment, a powdered composition is combined with aliquid carrier such as, i.e., water or a saline solution, with orwithout a stabilizing agent.

[0044] A specific formulation that may be used in the present inventionis a solution of lactoferrin in a hypotonic phosphate based buffer thatis free of potassium where the composition of the buffer is as follows:6 mM sodium phosphate monobasic monohydrate, 9 mM sodium phosphatedibasic heptahydrate, 50 mM sodium chloride, pH 7.0±0.1. Theconcentration of lactoferrin in a hypotonic buffer may range from 10microgram/ml to 100 milligram/ml. This formulation may be administeredvia any route of administration, for example, but not limited tointratumoral administration.

[0045] Further, a composition for topical administration which iscombined with a semi-solid carrier can be further formulated into a gelointment. A preferred carrier for the formation of a gel ointment is agel polymer. Preferred polymers that are used to manufacture a gelcomposition of the present invention include, but are not limited tocarbopol, carboxymethyl-cellulose, and pluronic polymers. Specifically,a powdered lactoferrin composition is combined with an aqueous gelcontaining an polymerization agent such as Carbopol 980 at strengthsbetween 0.5% and 5% wt/volume for application to the skin for treatmentof hyperproliferative disease on or beneath the skin.

[0046] Upon formulation, solutions are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective to result in an improvement or remediation ofthe symptoms. The formulations are easily administered in a variety ofdosage forms such as ingestible solutions, drug release capsules and thelike. Some variation in dosage can occur depending on the condition ofthe subject being treated. The person responsible for administrationcan, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations meetsterility, general safety and purity standards as required by FDA Officeof Biologics standards.

B. Treatment of Hyperproliferative Diseases

[0047] In accordance with the present invention, a lactoferrincomposition provided in any of the above-described pharmaceuticalcarriers is administered to a subject suspected of or having ahyperproliferative disease. One of skill in the art can determine thetherapeutically effective amount of human lactoferrin to be administeredto a subject based upon several considerations, such absorption,metabolism, method of delivery, age, weight, disease severity andresponse to the therapy.

[0048] The route of administration will vary, naturally, with thelocation and nature of the lesion, and include, for example intradermal,transdermal, parenteral, intravenous, intramuscular, intranasal,subcutaneous, percutaneous, intratracheal, intraperitoneal,intratumoral, perfusion, lavage, direct injection, and oraladministration.

[0049] Oral administration of the lactoferrin composition includes oral,buccal, enteral or intragastric administration. It is also envisionedthat the composition may be used as a food additive. For example, thecomposition is sprinkled on food or added to a liquid prior toingestion.

[0050] Intratumoral administration of the lactoferrin compositionincludes intratumoral injection, electroporation, or surgical orendoscopic implantation. Intratumoral injection, or injection into thetumor vasculature is specifically contemplated for discrete, solid,accessible tumors. Local, regional or systemic administration also maybe appropriate.

[0051] The hyperproliferative disease, includes but is not limited toneoplasms. A neoplasm is an abnormal tissue growth, generally forming adistinct mass that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias, lymphomas and myelomas. A tumor is the neoplasticgrowth of the disease cancer. As used herein, a “neoplasm”, alsoreferred to as a “tumor”, is intended to encompass hematopoieticneoplasms as well as solid neoplasms. Examples of neoplasms include, butare not limited to melanoma, non-small cell lung, small-cell lung, lung,hepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma, gum, tongue,leukemia, neuroblastoma, head, neck, breast, pancreatic, prostate,renal, bone, testicular, ovarian, mesothelioma, sarcoma, cervical,gastrointestinal, lymphoma, brain, colon, bladder, myeloma, or othermalignant or benign neoplasms.

[0052] Other hyperproliferative diseases include, but are not limited toneurofibromatosis, rheumatoid arthritis, Waginer's granulomatosis,Kawasaki's disease, lupus erathematosis, midline granuloma, inflammatorybowel disease, osteoarthritis, leiomyomas, adenomas, lipomas,hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis,pre-neoplastic lesions, carcinoma in situ, oral hairy leukoplakia, orpsoriasis, and pre-leukemias, anemia with excess blasts, andmyelodysplastic syndrome.

[0053] Particular neoplasms of interest in the present inventioninclude, but are not limited to hematopoietic neoplasms. For example, ahematopoietic neoplasm may include acute myelogenous leukemia, acutelymphoblastic leukemia, myelodysplastic syndrome, chronic myelomonocyticleukemia, juvenile myelomonocyte leukemia, multiple myeloma, chroniclymphocytic leukemia or other malignancy of hematologic origin.

[0054] In a preferred embodiment of the present invention, thelactoferrin compositions are administered in an effective amount todecrease, reduce, inhibit or abrogate the growth of a tumor. The amountmay vary from about 0.1 μg to about 100 g of the lactoferrincomposition. Preferably, the lactoferrin composition is orallyadministered in the range of 1 mg to 100 g per day, more preferablyabout 20 mg to about 10 g per day with the most preferred dose being 4.5g per day. Intravenously administered lactoferrin can be in the range of0.1 μg to about to 10 g per day, more preferably about 0.1 μg to about 1mg with the most preferred dose being 250 mg per day. Preferably, alactoferrin composition is intratumorally administered in the range of0.1 μg to 10 g per day with the most preferred dose being 100 μg perday. Topically, the amount of lactoferrin may vary from about 1 μg toabout 100 g of lactoferrin. Preferably, the topical gel, solution,capsule or tablet comprises a lactoferrin concentration of about 0.01%to about 20%. More preferably, the topical gel, solution, capsule ortablet may comprise a lactoferrin concentration of about 1% to about8.5%.

[0055] Treatment regimens may vary as well, and often depend on tumortype, tumor location, disease progression, and health and age of thepatient. Obviously, certain types of tumor will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

[0056] In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with the lactoferrin compositionmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional treatmentssubsequent to resection will serve to eliminate microscopic residualdisease at the tumor site.

[0057] Alternatively, the present invention may be used at the time ofsurgery, and/or thereafter, to treat residual or metastatic disease. Forexample, a resected tumor bed may be injected or perfused with aformulation comprising the lactoferrin composition. The perfusion may becontinued post-resection, for example, by leaving a catheter implantedat the site of the surgery. Periodic post-surgical treatment is alsoenvisioned.

[0058] Continuous administration also may be applied where appropriate,for example, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 6-12 hours, to about 12-24hours, to about 1-2 days, to about 1-2 weeks or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs. It was further contemplated that limbperfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

[0059] In specific embodiments, the lactoferrin composition is given ina single dose or multiple doses. The single dose may be administereddaily, or multiple times a day, or multiple times a week, or monthly ormultiple times a month. In a further embodiment, the lactoferrincomposition is given in a series of doses. The series of doses may beadministered daily, or multiple times a day, weekly, or multiple times aweek, or monthly, or multiple times a month.

[0060] A further embodiment of the present invention is a method oftreating a hyperproliferative disease comprising the step ofsupplementing a mucosal immune system by increasing the amount oflactoferrin in the gastrointestinal tract. Preferably, the lactoferrinis administered orally.

[0061] Still yet, a further embodiment is a method of enhancing amucosal immune response in the gastrointestinal tract in a subjectcomprising the step of administering orally to said subject alactoferrin composition, preferably human lactoferrin. It is envisionedthat lactoferrin stimulates interleukin-18 and GM-CSF in thegastrointestinal tract, which enhance immune cells. For example,interleukin-18 enhances T lymphocytes or natural killer cells and GM-CSFpromotes maturation and migration of immune cells including dendriticand other antigen presenting cells. In specific embodiments,interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is knownby those of skill in the art that IL-18 is a Th1 cytokine that acts insynergy with interleukin-12 and interleukin-2 in the stimulation oflymphocyte IFN-gamma production. Other cytokines may also be enhancedfor example, but not limited to IL-1b or, IL-12 or IFN-gamma. It is alsoenvisioned that lactoferrin stimulates interleukin-18 following oraladministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

[0062] A further embodiment of the present invention is a method oftreating a hyperproliferative disease comprising the step ofsupplementing the systemic immune system by increasing the amount oflactoferrin in the systemic circulation. Preferably, the lactoferrincomposition is administered intravenously. It is envisioned thatlactoferrin stimulates interleukin-18 and GM-CSF in the tissue, whichenhance immune cells. For example, interleukin-18 enhances T lymphocytesor natural killer cells and GM-CSF promotes maturation and migration ofimmune cells including dendritic and other antigen presenting cells. Inspecific embodiments, interleukin-18 (IL-18) enhances CD4+, CD8+ andCD3+ cells. It is known by those of skill in the art that IL-18 is a Th1cytokine that acts in synergy with interleukin-12 and interleukin-2 inthe stimulation of lymphocyte IFN-gamma production. Other cytokines mayalso be enhanced for example, but not limited to IL-1b or, IL-12 orIFN-gamma. It is also envisioned that lactoferrin stimulatesinterleukin- 18 following intravenous administration, which inhibitsangiogenesis and thereby has activity against tumor cells which aredependent on neovascularization.

[0063] A further embodiment of the present invention is a method oftreating a hyperproliferative disease comprising the step ofsupplementing a local or systemic immune system by increasing the amountof lactoferrin in the vicinity of the tumor. Vicinity of the tumorrefers to the general area of the tumor, for example the lactoferrin canbe administered directly into or on the tumor, or in the general area ofthe tumor, but not directly into the tumor. The general area may includethe margin area or near or adjacent the margin area of the tumor.Preferably, the lactoferrin composition is administered intratumorally.It is envisioned that lactoferrin stimulates interleukin-18 and GM-CSFin the local tissue, which enhances immune cells. For example,interleukin-18 enhances T lymphocytes or natural killer cells and GM-CSFpromotes maturation and migration of immune cells including dendriticand other antigen presenting cells. In specific embodiments,interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is knownby those of skill in the art that IL-18 is a Th1 cytokine that acts insynergy with interleukin-12 and interleukin-2 in the stimulation oflymphocyte IFN-gamma production. Other cytokines may also be enhancedfor example, but not limited to IL-1b or, IL12 or IFN-gamma. It is alsoenvisioned that lactoferrin stimulates interleukin-18 followingintratumoral administration, which inhibits angiogenesis and thereby hasactivity against tumor cells which are dependent on neovascularization.

[0064] A further embodiment of the present invention is a method oftreating a hyperproliferative disease comprising the step ofsupplementing a local or systemic immune system by increasing the amountof lactoferrin in the skin in the vicinity of the tumor. Preferably, thelactoferrin composition is administered topically. As above,administration in the vicinity of the tumor includes administration nearor adjacent to the margins of the tumor or directly in the margin areaof the tumor. It is envisioned that lactoferrin stimulatesinterleukin-18 and GM-CSF in the local tissue (e.g., keratinocytes),which enhances immune cells. For example, interleukin-18 enhances Tlymphocytes or natural killer cells and GM-CSF promotes maturation andmigration of immune cells including dendritic and other antigenpresenting cells. In specific embodiments, interleukin-18 (IL-18)enhances CD4+, CD8+ and CD3+ cells. It is known by those of skill in theart that IL-18 is a Th1 cytokine that acts in synergy withinterleukin-12 and interleukin-2 in the stimulation of lymphocyteIFN-gamma production. Other cytokines may also be enhanced for example,but not limited to EL-1b or, IL-12 or IFN-gamma. It is also envisionedthat lactoferrin stimulates interleukin-18 following intratumoraladministration, which inhibits angiogenesis and thereby has activityagainst tumor cells which are dependent on neovascularization.

C. Combination Treatments

[0065] In order to increase the effectiveness of the human lactoferrincomposition of the present invention, it may be desirable to combine thecomposition of the present invention with other agents effective in thetreatment of hyperproliferative disease, such as anti-cancer agents, orwith surgery. An “anti-cancer” agent is capable of negatively affectingcancer in a subject, for example, by killing cancer cells, inducingapoptosis in cancer cells, reducing the growth rate of cancer cells,reducing the incidence or number of metastases, reducing tumor size,inhibiting tumor growth, reducing the blood supply to tumor or cancercells, promoting an immune response against cancer cells or a tumor,preventing or inhibiting the progression of cancer, or increasing thelifespan of a subject with cancer. Anti-cancer agents include biologicalagents (biotherapy), chemotherapy agents, and radiotherapy agents. Moregenerally, these other compositions would be provided in a combinedamount effective to kill or inhibit proliferation of the cell. Thisprocess may involve administering the human lactoferrin composition ofthe present invention and the agent(s) or multiple factor(s) at the sametime. This may be achieved by administering a single composition orpharmacological formulation that includes both agents, or byadministering two distinct compositions or formulations, at the sametime, or at times close enough so as to result in an overlap of thiseffect, wherein one composition includes the human lactoferrincomposition and the other includes the second agent(s).

[0066] Alternatively, the lactoferrin composition of the presentinvention may precede or follow the other anti-cancer agent treatment byintervals ranging from minutes to weeks. In embodiments where the otheranti-cancer agent and lactoferrin composition are administered orapplied separately to the cell, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and lactoferrin composition would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one may contact the cellwith/administer both modalities within about 1-14 days of each otherand, more preferably, within about 12-24 hours of each other. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

1. Chemotherapy

[0067] Cancer therapies also include a variety of chemical basedtreatments. Some examples of chemotherapeutic agents include withoutlimitation antibiotic chemotherapeutics such as Doxorubicin,Daunorubicin, Adriamycin, Mitomycin (also known as mutamycin and/ormitomycin-C), Actinomycin D (Dactinomycin), Bleomycin, Plicomycin, plantalkaloids such as Taxol, Vincristine, Vinblastine, miscellaneous agentssuch as platinum based agents (e.g., Cisplatin (CDDP)), etoposide(VP16), Tumor Necrosis Factor, and alkylating agents such as,Carmustine, Melphalan (also known as alkeran, L-phenylalanine mustard,phenylalanine mustard, L-PAM, or L-sarcolysin, (a phenylalaninederivative of nitrogen mustard), Cyclophosphamide, Chlorambucil,Busulfan (also known as myleran), taxane based agents (e.g., docetaxel)and Lomustine.

[0068] Some examples of other agents include, but are not limited to,Carboplatin, Procarbazine, Mechlorethamine, Irinotecan, Topotecan,Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen, Raloxifene,Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, ICI182,780, EM-800, Estrogen Receptor Binding Agents, Gemcitabinen,Navelbine, Farnesyl-protein transferase inhibitors, Transplatinum,5-Fluorouracil, hydrogen peroxide, and Methotrexate, Temazolomide (anaqueous form of DTIC), Mylotarg, Dolastatin-10, Bryostatin, or anyanalog or derivative variant of the foregoing.

2. Radiotherapeutic Agents

[0069] Radiotherapeutic agents and factors include radiation and wavesthat induce DNA damage for example, γ-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions, radioisotopes, and thelike. Therapy may be achieved by irradiating the localized tumor sitewith the above described forms of radiations. It is most likely that allof these factors effect a broad range of damage to DNA, the precursorsof DNA, the replication and repair of DNA, and the assembly andmaintenance of chromosomes.

[0070] Dosage ranges for X-rays range from daily doses of 50 to 200roentgens for prolonged periods of time (3 to 4 weeks), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

3. Surgery

[0071] Approximately 60% of persons with cancer will undergo surgery ofsome type, which includes preventative, diagnostic or staging, curativeand palliative surgery. Curative surgery is a cancer treatment that maybe used in conjunction with other therapies, such as the treatment ofthe present invention, chemotherapy, radiotherapy, hormonal therapy,gene therapy, immunotherapy and/or alternative therapies.

[0072] Curative surgery includes resection in which all or part ofcancerous tissue is physically removed, excised, and/or destroyed. Tumorresection refers to physical removal of at least part of a tumor. Inaddition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and miscopically controlledsurgery (Mohs' surgery). It is further contemplated that the presentinvention may be used in conjunction with removal of superficialcancers, precancers, or incidental amounts of normal tissue.

[0073] Upon excision of part of all of cancerous cells, tissue, ortumor, a cavity may be formed in the body. Treatment may be accomplishedby perfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

4. Other Biotherapy Agents

[0074] It is contemplated that other biological agents may be used incombination with the present invention to improve the therapeuticefficacy of treatment. These additional agents include, withoutlimitation, agents that affect the upregulation of cell surfacereceptors and GAP junctions, cytostatic and differentiation agents,inhibitors of cell adhesion, agents that increase the sensitivity of thehyperproliferative cells to apoptotic inducers, or other biologicalagents, as well as biotherapy such as for example, hyperthermia.

[0075] Hyperthermia is a procedure in which a patient's tissue isexposed to high temperatures (up to 106° F). External or internalheating devices may be involved in the application of local, regional,or whole-body hyperthermia. Local hyperthermia involves the applicationof heat to a small area, such as a tumor. Heat may be generatedexternally with high-frequency waves targeting a tumor from a deviceoutside the body. Internal heat may involve a sterile probe, includingthin, heated wires or hollow tubes filled with warm water, implantedmicrowave antennae, or radiofrequency electrodes.

[0076] A patient's organ or a limb is heated for regional therapy, whichis accomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

[0077] Hormonal therapy may also be used in conjunction with the presentinvention. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen and this often reduces the risk of metastases.

[0078] Adjuvant therapy may also be used in conjunction with the presentinvention. The use of adjuvants or immunomodulatory agents include, butare not limited to tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; orMIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines.

5. Immunotherapy

[0079] Immunotherapeutics, generally, rely on the use of immune effectorcells and molecules to target and destroy cancer cells. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually effect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells.

[0080] It is contemplated that vaccines that are used to treat cancermay be used in combination with the present invention to improve thetherapeutic efficacy of the treatment. Such vaccines include peptidevaccines or dendritic cell vaccines. Peptide vaccines may include anytumor-specific antigen that is recognized by cytolytic T lymphocytes.Yet further, one skilled in the art realizes that dendritic cellvaccination comprises dendritic cells that are pulsed with a peptide orantigen and the pulsed dendritic cells are administered to the patient.

[0081] Examples of tumor-specific antigens that are being used asvaccines in melanoma include, but are not limited to gp100 or MAGE-3.These antigens are being administered as peptide vaccines and/or asdendritic cell vaccines.

D. EXAMPLES

[0082] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Inhibition of Tumor Growth by rhLF

[0083] Human squamous cell carcinoma (O12) was used. The cells wereinjected into the right flank of athymic nude mice. rhLF wasadministered either intratumorally (49 animals, 7 doses ranging from0.05 μg to 125 μg per dose), intravenously (7 animals, 125 ug/dose) ororally (7 animals 20 mg/dose). Control animals were treated with onlythe vehicle; no rhLF was administered to the control animals. rhLF wasadministered twice a day for either five days (intravenous group) oreight days (all other groups) starting 11 days after inoculation withtumor cells to allow forrnation of established tumors.

[0084] The efficacy of treatment was evaluated by measuring the solidtumor size during and at the end of the experiment; the body weightswere also determined at the time of tumor measurements. As seen in FIG.1 and Table 1, treatment with rhLF reduced rates of tumor growthrelative to the control by 46% to 80%. In fact, oral treatment with 20mg rhLF most significantly reduced the tumor growth, by 80% compared tothe control (p=0.0073). TABLE 1 Summary of tumor growth inhibition byrhLF in O12 tumor model in mice Inhibition Relative to Placebo P valueDay 19* Day 28* Day 19* Day 28* Intratumoral 28% 46% 0.139 0.0263** N =49 Intravenous 55% 65% 0.0666 0.0233** N = 7 Oral 76% 80% 0.0175**0.0073*** N = 7

[0085] Results from this study showed that rhLF administered by multipleroutes significantly inhibited tumor growth in a squamous cell tumormodel in mice, with oral administration being the most effective. Basedupon these results, it was further contemplated that oral lactoferrinaffects the tumor by enhancing immune cell activity.

Example 2 Evaluation of rhLF in Tumor Types

[0086] Tumor cells from a broad range of tumor types are injected intothe right flank of athymic nude mice. Animals are administered eitherrhLF, native hLF or bovine LF orally. Control animals are treated withonly the vehicle, no rhLF is administered to the control animals. rhLFis administered either once or twice a day for either one, five, sevenor fourteen days or eight days starting approximately eleven days afterinoculation with tumor cells to allow formation of established tumors orat such other time as is generally done with standard or publishedregimens.

[0087] The efficacy of treatment is evaluated by measuring the solidtumor size during and at the end of the experiment; the body weights arealso determined at the time of tumor measurements. The immune responseis measured by measuring the amount of cytokines, T-cells and NK cellsin circulation and in the intestine.

Example 3 Effect of Oral Administration of rhLF and bLF

[0088] Recombinant human lactoferrin and bovine lactoferrin were orallyadministered to mice, and the production of IL-18 in the small intestinewas measured.

[0089] Mice were treated for three days daily with 65 mg/kg/day of rhLF,300 mg/kg/day of rhLF or 300 mg/kg/day of bLF. For a control, mice wereonly administered the pharmaceutical carrier. Twenty-four hoursfollowing administration of the LF or control for 3 days, animals wereweighed and blood and serum were collected. Serum was used for cytokineELISA assays.

[0090] Also, at these time points, animals were sacrificed and the smallintestinal tissue was removed for further analysis. Small intestinalepithelium was homogenized using a lysis buffer consisting of PBS, 1%Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphatecontaining 10 μg/ml PhenylMetheylsulfonyl fluoride. Homogenate wascentrifuiged at 15,000 rpm for 10 minutes and the supernatant stored at−80 C. till it was tested for IL-18 levels.

[0091] As seen in Table 2 and Table 3, administration of rhLF at bothdoses significantly enhanced the amounts of IL-18 in both the serum andin the intestinal extract. Bovine LF caused a lesser increase in theintestinal IL-18 levels and did not increase the serum levels of IL-18.TABLE 2 Effect of rhLF and bLF on IL-18 levels in the gut and serumIntestinal Extract (pg) Serum (pg) Control 955 141 300 mg/kg bLF 4,515134  65 mg/kg rhLF 7,879 259 300 mg/kg rhLF 8,350 328

[0092] TABLE 3 Stimulation by rhLF and bLF of IL-18 levels in the gutand serum Intestinal Extract Serum % Increase P-value % Increase P-valueIncrease Over Control 300 mg/kg bLF 373% 0.0086  −5% 0.5411  65 mg/kgrhLF 725% 0.0034    84% 0.0132 300 mg/kg rhLF 775% 0.0001   132% 0.0007Increase Over Blf  65 mg/kg rhLF  75% 0.1490    94% 0.0366 300 mg/kgrhLF  85% 0.0617   145% 0.0084

Example 4 Effect of Oral rhLF on NK Activity in vivo

[0093] Balb/c naive mice were treated orally with rhLF or placebo once aday for 3 days (see Table 4). TABLE 4 Treatment Regimen Treatment* NDose (mg/kg) Route Schedule Group 1 Placebo 6 0 — — Group 2 RhLF 7 300mg/kg/day Oral 3 days

[0094] On day 4, mice were sacrificed and spleens were collected. NKcells were separated using a magnetic bead cell sorting assay (MACSanti-NK-DX5) and counted. Cells were then tested in vitro forNK-activity against YAC targets using a lactate dehydrogenase (LDH)release test.

[0095] Table 5 shows that oral rhLF treatment resulted in a significantincrease of NK activity ex-vivo against YAC-target cells (10%@30:1versus 2.8% of ctrl group). No significant change in NK activity wasobserved in placebo treated mice. TABLE 5 NK activity in mice treatedwith oral rhLF Control Low Medium High 0.056 0.09 0.407 PlaceboRhLF-treated Raw data Raw data E:T % Cyto- E:T % Cytotoxicity* E:T cellE cell toxicity* cell E cell Increased ratio mix ctrl Final mix ctrlFinal over ctrl 30:1 0.281 0.215 2.86 0.358 0.267 9.81 7** 15:1 0.1760.110 2.85 0.214 0.143 4.12 1.3  7.5:1 0.117 0.054 2.21 0.131 0.074 0.440  3.7:1 0.086 0.030 0.19 0.096 0.042 0 0 

Example 5 Effect of Oral Administration of rhLF on GM-CSF in vivo

[0096] Recombinant human lactoferrin or placebo were orally administeredto mice, and the production of GM-CSF in the small intestine wasmeasured.

[0097] Mice (5 animals per group) were treated for three days daily with300 mg/kg/day of rhLF. For a control, mice were only administered thepharmaceutical carrier. Twenty-four hours following administration ofthe LF or placebo for 3 days, animals were and the small intestinaltissue was removed for further analysis. Small intestinal epithelium washomogenized using a lysis buffer consisting of PBS, 1% Nonidet P-40,0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate containing 10μg/ml PhenylMetheylsulfonyl fluoride. Homogenate was centrifuged at15,000 rpm for 10 minutes and the supernatant stored at −80 C. till itwas tested for GM-CSF levels using an ELISA kit.

[0098] As shown in Table 6, treatment with rhLF increased the productionof a key immunostimulatory cytokine, GM-CSF, in the small intestinerelative to the placebo treated animals. TABLE 6 Effect of rhLF onGM-CSF levels in the gut and serum Mean (SEM) in pg Increase overPlacebo Placebo 6.48 (0.32) — 300 mg/kg rhLF 7.74 (0.19) 19.4% (p <0.01)

Example 6 Combination of hLF with Chemotherapy

[0099] A murine squamous carcinoma cell line (SCCVII) was injected intothe floor of the mouth through the neck skin of immunocompetent C3H mice(Day 0). Five days after tumor cell implantation (Day 5), a skinincision was made in the lower neck and surgical dissection revealed theestablished tumors. Tumors were measured in three dimensions withcalipers.

[0100] Tumor-bearing mice were randomized into a control group and sevengroups receiving rhLF and/or cisplatin. RhLF (4 mg; 200 mg/Kg) wasadministered once daily by oral gavage for 8 days on days 5 through 12.Cisplatin was administered as a single dose of 5 mg/Kg givenintraperitoneally either at the start of rhLF (day 5), in the middle(day 8) or at the end (day 12) of rhLF therapy. Animals were sacrificedon day 12 post-implantation and the residual tumor masses were measuredand processed for later additional analyses. TABLE 7 Experimental GroupsDescription N RhLF mg/kg Cisplatin Group A Placebo 5 0 (Placebo) 0 GroupB RhLF Alone 5 200 mg/kg 0 Group C CP Day 5 5 0 5 mg/kg on day 5* GroupD CP Day 8 5 0 5 mg/kg on day 8* Group E CP Day 12 4 0 5 mg/kg on day 12Group F RhLF/CP-5 5 200 mg/kg 5 mg/kg on day 5* Group G RhLF/CP-8 5 200mg/kg 5 mg/kg on day 8* Group H RhLF/CP-12 5 200 mg/kg 5 mg/kg on day12*

[0101] Mice treated either with rhLF alone, cisplatin alone or bothagents, showed a tumor growth inhibition (TGI) relative to the placeboanimals. The maximum inhibition was observed in the group receiving boththerapies (Table 7 and FIG. 2).

[0102] In all cases, the animals receiving rhLF+cisplatin showed a TGIrelative to the relevant group receiving cisplatin alone. When pooledfor analysis, animals receiving rhLF+cisplatin showed a 77% TGI relativeto the placebo animals (P<0.0001), a 66% TGI relative to rhLF alone(P<0.01) and a 63% TGI relative to cisplatin alone (P<0.01).

[0103] Cisplatin dosing immediately prior to the start of rhLF(RhLF+CP-5) or during the period of rhLF administration (RhLF+CP-8)provided greater incremental benefit than when cisplatin wasadministered following completion of rhLF therapy (RhLF+CP-12). However,only the straddling regimen (RhLF+CP-8) provided a statisticallysignificant improvement (P<0.01) TGI of 77% over cisplatin alone (CP Day8). TABLE 8 Tumor Growth Inhibition (TGI) by Treatment Group GrowthRelative to Placebo* Group (SEM) TGI (%) P-value A (Placebo) 741 (79) —— B (RhLF alone) 496 (155) 33% 0.0989 C (CP Day 5) 240 (137) 68% 0.0066D (CP Day 8) 693 (146)  6% 0.3898 E (CP Day 12) 433 (175) 42% 0.0634 F(RhLF + CP-5)  14 (5) 98% <0.0001 G (RhLF + CP-8) 159 (48) 79% 0.0001 H(RhLF + CP-12) 331 (47) 55% 0.0011 C to E (All CP) 457 (96) 38% 0.0564 Fto H (All rhLF/CP) 168 (40) 77% <0.0001

Example 7 Dose Dependence of Combining hLF with Chemotherapy

[0104] A murine squamous carcinoma cell line (SCCVII) was injected intothe floor of the mouth through the neck skin of immunocompetent C3H mice(Day 0) as described in Example 6. On Day 5 days after initialimplantation, tumors were measured for the baseline, then treated witheither cisplatin (Day 8, i.p., 5 mg/kg) alone or cisplatin plus threedoses of oral rhLF (daily by gavage for 7-8 days on days 5 through11/12). Animals were sacrificed on Day 11/12 and tumors measured. Therewas a dose dependent inhibition of tumor growth in the animals receivingboth rhLF and cisplatin as compared to the animals receiving cisplatinalone as shown in FIG. 3.

Example 8 Combination of hLF with Docetaxel

[0105] A murine squamous carcinoma cell line (SCCVII) was injected intothe floor of the mouth through the neck skin of immunocompetent C3H mice(Day 0) as described in Example 7. On Day 5 after initial implantation,tumors were measured for the baseline, then treated with either oralplacebo alone (once daily from days 5 to 12; 6 animals), placebo anddocetaxel (i.v. bolus of 31.3 mg/kg docetaxel on Day 8; 9 animals), ordocetaxel plus oral rhLF (200 mg/kg, administered once daily by gavagefrom days 5 to 12; 9 animals). Animals were sacrificed on Day 14 andtumors measured. Docetaxel alone caused an inhibition of tumor growthrelative to placebo and the combination of rhLF and docetaxel induced afurther growth inhibition. Inhibition and p-values (1-tailed) are shownin Table 9. TABLE 9 Tumor Growth Inhibition (TGI) by Treatment GroupGrowth Relative to Placebo* Relative to Docetaxel Group (SEM) TGI (%)P-value TGI (%) P-value Placebo 5,157 (497) — — — — Docetaxel 2,103(209) 59% <0.0001 — — Docetaxel + 1,288 (286) 75% <0.0001 39% 0.0175rhLF

Example 9 Combination of hLF with Radiotherapy

[0106] A murine squamous carcinoma cell line (SCCVI) was injected intothe floor of mouth through the neck skin of immunocompetent C3H mice(Day 0). Five days after tumor cell implantation (Day 5), a skinincision was made in the lower neck and surgical dissection revealed theestablished tumors. Tumors were measured in three dimensions withcalipers.

[0107] Tumor-bearing mice were randomized into six groups receiving rhLF(200 mg/Kg) and/or radiotherapy as described below. RhLF (4 mg; 200mg/kg) was administered by oral gavage once daily for 8 days on days 5through 12. Radiotherapy was administered as single dose of 2 Gray givenat the begirning (day 5) or at during (day 8) rhLF-therapy. Animals weresacrificed on day 14 post-treatment and the residual tumor masses weremeasured and processed for later additional analyses. TABLE 10Experimental Groups Description N RhLF mg/kg* Radiation Group A Placebo10 0 (Placebo) None Group B RhLF Alone 10 200 mg/kg None Group CRadiation Day 5  8 0 2 Gray on day 5 Group D RhLF/Rad 5 10 200 mg/Kg 2Gray on day 5 Group E Radiation Day 8 10 0 2 Gray on day 8 Group FRhLF/Rad 8 10 200 mg/kg 2 Gray on day 8

[0108] Mice receiving rhLF alone, radiotherapy alone, or combinationtherapy showed a significant tumor growth inhibition (TGI) relative toplacebo treated mice. The mice receiving both rhLF and radiation showeda modest increase in TGI over monotherapy with rhLF (28%, P<0.05) andradiation (15%, P=0.1207). TABLE 11 Tumor Growth Inhibition (TGI) byTreatment Group: Growth Relative to Placebo Group (SEM) Inhibition*P-value* A (Placebo) 2348 (395) — B (rhLF alone) 1074 (163) 54% 0.0040 C(Radiation Day 5)  827 (105) 65% 0.0021 D (rhLF/Rad 5)  750 (125) 68%0.0006 E (Radiation Day 8)  977 (112) 58% 0.0018 F (rhLF/Rad 8)  797(119) 66% 0.0007 C/E (Both Radiation)  911 (78) 61% <0.0001 D/F (BothrhLF/Rad)  774 (84) 67% <0.0001

[0109] Thus, lactoferrin stimulated the immune system. Still further,lactoferrin in combination with cisplatin, docetaxel and radiationresulted in inhibition of tumor growth.

Example 10 Oral Administration of hLF in Humans

[0110] Recombinant human lactoferrin was orally administered to humanpatients with a range of metastatic cancer types that had failedstandard chemotherapy in two different studies conducted in multiplecenters in four countries (Argentina, Brazil, Chile, U.S.) RhLF wasadministered at doses of 1.5 to 9 grams daily in two divided doses incycles of 14 each with a 14 day gap.

[0111] Tumor size progression was monitored through CT scans and tumormarkers where available. CT scans were performed at baseline and aftereach 8-week period once treatment was initiated, and also compared witha pre-baseline scan conducted prior to enrollment in the study. Tumormarkers are measured every 4 weeks. Blood samples were collected tomeasure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples were collected to measurecirculating IL-18, IL-1, IL-2, and IL-4, IL-5, IL-10, IL-12 and IFN-γ.

[0112] Out of nineteen evaluable patients (those with a baseline CT scanand at least one post-treatment scan), nine patients (47%) exhibitedstable disease by the RECIST criteria at the time of the firstpost-treatment scan. Patients with a broad range of tumor types showed abenefit from lactoferrin administration.

[0113] Table 12 shows the tumor response of five individual patientswith different tumor types. In all cases, the percent growth of thetumor size prior to treatment of rhLF (the relevant duration of time isshown in parentheses) and the growth of the tumor in the ensuing twotime periods, as measured by CT, showed a diminution in their rate oftumor growth or an actual shrinkage. TABLE 12 Tumor Response of PatientsReceiving Oral rhLF for treatment of Metastatic Cancer Pretreatment PostTreatment 1 Post Treatment 2 % Growth % Growth % Growth Patient# Cancer(Weeks) (Weeks) (Weeks) # 204 Breast  40% (8)    0% (10)    0% (6.5) #106 Melanoma  24% (19) −18% (11) Not yet done # 104 Gastric  25% (5.5)  10% (10)  −5% (7) # 102 Ovarian  30% (21)  −5% (10.5)  −7% (8.5) # 007Lung 160% (5.5)   13% (7.5)   12% (8.5)

Example 11 Combination Therapy with Oral hLF in Humans

[0114] Recombinant human lactoferrin is orally administered to humanpatients to inhibit tumor growth either alone or in combination withstandard anti-cancer regimens.

[0115] Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 10 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

[0116] Tumor size progression is monitored through CT scans and tumormarkers where available. CT scans are performed at baseline and aftereach 8-week period once treatment is initiated. Tumor markers aremeasured every 4 weeks once treatment is initiated. Blood samples arecollected to measure subclasses of circulating lymphocytes and NK cellactivity. Plasma, serum and blood cell extract samples are collected tomeasure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, and IL-12 andIFN-γ.

Example 12 Immunostimulation Following Administration of Oral rhLF

[0117] Balb/c naïve mice were treated orally with rhLF or placebo once aday for 3 days. One day later (day 4), mice were sacrificed and spleenscollected. NK cells were separated using a magnetic bead cell sortingassay (MACS anti-NK-DX5) and counted. Cells were then tested in vitrofor NK-activity against YAC targets using a lactate dehydrogenase (LDH)release test.

[0118] As shown in FIG. 4, oral rhLF treatment resulted in a significantincrease of NK activity ex-vivo against YAC-target cells. At a 30:1 E:Tratio rhLF administration resulted in a 243% relative increase overplacebo-treated animals (from 2.86% to 9.81%; p<0.05).

Example 13 Effect of Intravenous Administration

[0119] Recombinant lactoferrin, bovine lactoferrin and nativelactoferrin are intravenously administered to animals, preferably rats,and the production of IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 andIFN-gamma in the plasma, serum and blood packed cells are measured.

[0120] Briefly, rats are treated for fourteen consecutive days with 0.05μg to 1000 μg per dose. For a control, rats are only administered thepharmaceutical carrier. At specific time points following administrationof the LF or control for 0 days, 2 days, 3 days, 5 days, 9 days and 14days, animals are weighed and blood and serum are collected. The levelsof CD4+, CD8+ and NK cells are counted from the blood that wascollected. Plasma, serum and an extract of the blood cells are used forcytokine ELISA assays.

[0121] Also, at 24 day time point, animals are sacrificed and tissuesare removed for further analysis. Tissues are homogenized using a lysisbuffer consisting of PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and0.1% sodium dodecyl sulphate containing 10 μg/ml PhenylMetheylsulfonylfluoride. Homogenate is centrifuged at 15,000 rpm for 10 minutes and thesupernatant stored at −80 C. till it is tested for the cytokines IL-18,IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-gamma.

Example 14 Combination Chemotherapy of Intravenously rhLF with otherAgents

[0122] Tumor cells to be tested are injected into the right flank ofathymic nude mice. Animals are administered rhLF intravenously alone andin combination with other anti-cancer regimens as described in Example13. Control animals are treated with only the vehicle; no rhLF isadministered to the control animals. rhLF is administered using regimensidentified as being optimal in the trials described in Example 13.Anti-cancer therapy is administered using standard or publishedregimens. Therapy starts approximately 11 days after inoculation withtumor cells to allow formation of established tumors or at such othertime as is generally done with standard or published regimens.

[0123] The efficacy of individual and combination treatments areevaluated by measuring the solid tumor size during and at the end of theexperiment; the body weights are also determined at the time of tumormeasurements.

Example 15 Intravenous Administration of hLF in Humans

[0124] Recombinant lactoferrin is intravenously administered to patientsto inhibit tumor growth.

[0125] Briefly, rhLF at a dose of 500 mg per day for eight days topatients suffering from unresectable or metastatic cancer.Alternatively, rhLF is administered for one to eight days to patientssuffering from metastatic cancer in daily doses of 0.1, 1, 10, 100, and1000 mg. The dose is administered intravenously.

[0126] Tumor size progression is monitored through CT scans and tumormarkers where available. CT scans are performed at baseline and aftereach 8-week period once treatment is initiated. Tumor markers aremeasured every 4 weeks. Blood samples are collected to measuresubclasses of circulating lymphocytes and NK cell activity. Plasma,serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 16 Combination Therapy with Intravenous hLF

[0127] Recombinant lactoferrin is intravenously administered to patientsto inhibit tumor growth either alone or in combination with standardanti-cancer regimens.

[0128] Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 15 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

[0129] Tumor size progression is monitored through CT scans and tumormarkers where available. CT scans are performed at baseline and aftereach 8-week period once treatment is initiated. Tumor markers aremeasured every 4 weeks once treatment is initiated. Blood samples arecollected to measure subclasses of circulating lymphocytes and NK cellactivity. Plasma, serum and blood cell extract samples are collected tomeasure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 andIFN-γ.

Example 17 Activity of Intratumoral rhLF

[0130] O12 human oropharyngeal squamous cell carcinoma tumor cells wereinjected to the right flank of athymic nude mice. Recombinant humanlactoferrin and vehicle controls were dosed via intratumoral injection.Each animal was administered different concentrations of rhLF in 50 μLdoses consisting of four separate injections of approximately 12.5 μL ofthe dose, at different directions and angles (approximately S/N/E/W) toensure that the dose was distributed evenly throughout the tumor(fanning). TABLE 13 Treatment schedule of intratumor injections ofrecombinant human lactoferrin in O12 human squamous carcinoma celltumors in nude mice Dose of rhLF per animal in group Group Regimen 0 1 23 4 5 E. A Once on day 1, 0 100 μg 250 μg 500 μg 250 na kill 8 dayslater μg* (nude mice) C Twice/day for 0  25 μg  50 μg 125 μg 250 500 8days starting μg  μg on Day 11 after inoculation, kill on Day 20 (nudemice)

[0131] Table 13 shows the regimen followed for each experimental groupand the dose of rhLF per injection for each animal per group. In thisstudy, rhLF was administered directly into the tumor. Each animal wastracked daily for tumor growth by external caliper measurements of theprotruding tumor.

[0132] Using this model, significant reduction of tumor growth wasevident in both rhLF treated groups relative to the control animals.Compared to the median tumor size for the pooled placebo samples fromgroups A and C, the rates of tumor growth in animals receiving a singledose of rhLF (Group A) were reduced by 50% on day 11 after theadministration of rhLF (p<0.05). The rates of tumor growth in animalsdosed twice daily (Group C) were reduced by 56% when compared to thepooled control group (p<0.01) (See FIG. 5).

Example 18 Immune Stimulation Following Intratumoral rhLF

[0133] Normal C3H/HeJ mice were implanted with one of two mouse tumorsfollowing the methodology described in Example 17. Tumors used wereSCCVII and RIF mouse tumor cell lines. Following establishment of thetumors in the mice, tumors were injected intratumorally daily for 4 dayswith 250 or 500 μg rhLF per dose or with vehicle control. Twenty fourhours following the last intratumoral injection, animals were sacrificedand the blood examined for lymphocyte populations. The number ofcirculating lymphocytes were increased by 34% to 56% relative to theplacebo treated control animals (Table 14). TABLE 14 Increase incirculating lymphocytes following intratumoral administration of rhLFCD3+ CD4+ CD8+ Number of Cells Placebo 2104 1800  785 rhLF treated 32912621 1054 Increase with rhLF 56% 46% 34%

Example 19 Combination Chemotherapy of hLF with other Agents

[0134] Tumor cells to be tested are injected into the right flank ofathymic nude mice. Animals are administered rhLF intratumorally aloneand in combination with other anti-cancer regimens as described inExample 1 or Example 17. Control animals are treated with only thevehicle; no rhLF is administered to the control animals. Anti-cancertherapy is administered using standard or published regimens. Therapystarts approximately 11 days after inoculation with tumor cells to allowformation of established tumors or at such other time as is generallydone with standard or published regimens.

[0135] The efficacy of individual and combination treatments areevaluated by measuring the solid tumor size during and at the end of theexperiment; the body weights are also determined at the time of tumormeasurements.

Example 20 Intratumoral Administration of hLF

[0136] Recombinant lactoferrin is intratumorally administered topatients to inhibit tumor growth.

[0137] Briefly, rhLF at a dose of 1000 μg per day for eight days topatients suffering from unresectable or metastatic cancer.Alternatively, rhLF is administered for one to eight days to patientssuffering from metastatic cancer in daily doses of 10, 50, 100, 500 and1000 μg. The dose is administered intratumorally.

[0138] Tumor size progression is monitored through CT scans and tumormarkers where available. CT scans are performed at baseline and aftereach 8-week period once treatment is initiated. Tumor markers aremeasured every 4 weeks. Blood samples are collected to measuresubclasses of circulating lymphocytes and NK cell activity. Plasma,serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 21 Combination Therapy with Intratumoral hLF

[0139] Recombinant lactoferrin is intratumorally administered topatients to inhibit tumor growth either alone or in combination withstandard anti-cancer regimens.

[0140] Briefly, rhLF is administered using the optimum regimen and dosesidentified in Example 20 and the standard anti-cancer regimen(s) for theselected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

[0141] Tumor size progression is monitored through CT scans and tumormarkers where available. CT scans are performed at baseline and aftereach 8-week period once treatment is initiated. Tumor markers aremeasured every 4 weeks once treatment is initiated. Blood samples arecollected to measure subclasses of circulating lymphocytes and NK cellactivity. Plasma, serum and blood cell extract samples are collected tomeasure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 andIFN-γ.

Example 22 Topical Administration of hLF in Humans

[0142] Recombinant lactoferrin in a gel formulation is administered topatients to inhibit tumor growth.

[0143] Briefly, rhLF gel at strengths of 1%, 2.5% or 8.5% is appliedtwice a day to a skin or subcutaneous cancerous lesion in a patient withmetastatic disease. Application of rhLF gel continues till tumorprogression.

[0144] Size progression of the metastatic disease is monitored throughCT scans and tumor markers where available. CT scans are performed atbaseline and after each 8-week period once treatment is initiated. Tumormarkers are measured every 4 weeks. Blood samples are collected tomeasure subclasses of circulating lymphocytes and NK cell activity.Plasma, serum and blood cell extract samples are collected to measurecirculating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-γ.

Example 23 Combination Therapy with Topical hLF

[0145] Recombinant lactoferrin in a gel formulation is administered topatients to inhibit tumor growth either alone or in combination withstandard anti-cancer regimens.

[0146] Briefly, rhLF is administered using the optimum regimen and dosesidentified in Examples 22 and the standard anti-cancer regimen(s) forthe selected tumor type is used as part of the combination therapy. Theroute of administration and regimen of the additional anti-cancertherapy is as approved by the FDA for that indication or as described ina peer reviewed publication.

[0147] Size progression of the metastatic disease is monitored throughCT scans and tumor markers where available. CT scans are performed atbaseline and after each 8-week period once treatment is initiated. Tumormarkers are measured every 4 weeks once treatment is initiated. Bloodsamples are collected to measure subclasses of circulating lymphocytesand NK cell activity. Plasma, serum and blood cell extract samples arecollected to measure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10,IL-12 and IFN-γ.

References Cited

[0148] All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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[0178] Although the present invention and its advantages have beendescribed in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe spirit and scope of the invention as defined by the appendeddescription. Moreover, the scope of the present application is notintended to be limited to the particular embodiments of the process,machine, manufacture, composition of matter, means, methods and stepsdescribed in the specification. As one of ordinary skill in the art willreadily appreciate from the disclosure of the present invention,processes, machines, manufacture, compositions of matter, means,methods, or steps, presently existing or later to be developed thatperform substantially the same function or achieve substantially thesame result as the corresponding embodiments described herein may beutilized according to the present invention. Accordingly, the appendeddescriptions are intended to include within their scope such processes,machines, manufacture, compositions of matter, means, methods, or steps.

What is claimed is:
 1. A method of treating a hyperproliferative diseasecomprising the step of administering intratumorally to a subject alactoferrin composition in an amount sufficient to provide animprovement in the hyperproliferative disease in said subject.
 2. Themethod of claim 1, wherein said lactoferrin composition is dispersed ina pharmaceutically acceptable carrier.
 3. The method of claim 1, whereinsaid lactoferrin is mammalian lactoferrin.
 4. The method of claim 3,wherein said lactoferrin is human.
 5. The method of claim 3, whereinsaid lactoferrin is bovine.
 6. The method of claim 3, wherein saidlactoferrin is recombinant lactoferrin.
 7. The method of claim 1,wherein the amount of the lactoferrin composition that is administeredis about 0.1 μg to about 10 g per day.
 8. The method of claim 1, whereinthe hyperproliferative disease is further defined as cancer.
 9. Themethod of claim 8, wherein the cancer comprises a neoplasm.
 10. Themethod of claim 9, wherein the neoplasm is selected from the groupconsisting of melanoma, non-small cell lung, small-cell lung, lunghepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma, leukemia,neuroblastoma, squamous cell, head, neck, gum, tongue, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,sarcoma, cervical, gastrointestinal, lymphoma, brain, colon, andbladder.
 11. The method of claim 1, wherein the hyperproliferativedisease is selected from the group consisting of rheumatoid arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,atherosclerosis, pre-neoplastic lesions, carcinoma in situ, oral hairyleukoplakia, and psoriasis.
 12. A method of treating ahyperproliferative disease comprising the step of supplementing asystemic immune system in a subject by increasing the amount oflactoferrin at the site of the hyperproliferative disease.
 13. Themethod of claim 12, wherein said lactoferrin is recombinant lactoferrin.14. The method of claim 12, wherein said lactoferrin is administeredintratumorally.
 15. The method of claim 12, wherein said lactoferrinstimulates the production of interleukin-18.
 16. The method of claim 12,wherein said lactoferrin stimulates the production of GM-CSF.
 17. Amethod of treating a hyperproliferative disease comprising the step ofsupplementing the local immune system in a subject by increasing theamount of lactoferrin at the site of the hyperproliferative disease. 18.The method of claim 17, wherein said lactoferrin is recombinantlactoferrin.
 19. The method of claim 17, wherein said lactoferrin isadministered intratumorally.
 20. The method of claim 17, wherein saidlactoferrin stimulates the production of interleukin-18.
 21. The methodof claim 20, wherein interleukin-18 stimulates the production,maturation or activity of immune cells.
 22. The method of claim 17,wherein said lactoferrin stimulates the production of GM-CSF.
 23. Amethod of enhancing a local immune response in the vicinity of a tumorfollowing the step of administering intratumorally to said subject alactoferrin composition.
 24. The method of claim 23, wherein saidlactoferrin is recombinant lactoferrin.
 25. The method of claim 23,wherein said lactoferrin composition stimulates interleukin-18 in thesite of injection.
 26. The method of claim 23, wherein said lactoferrincomposition stimulates GM-CSF in the site of injection.
 27. The methodof claim 25, wherein interleukin-18 stimulates the production,maturation or activity of immune cells.
 28. The method of claim 27,wherein the immune cells are T lymphocytes or natural killer cells. 29.The method of claim 28, wherein the T lymphocytes are selected from thegroup consisting of CD4+, CD8+ and CD3+ cells.
 30. The method of claim26, wherein said GM-CSF stimulates the production, maturation oractivity of immune cells.
 31. The method of claim 30, wherein the immunecells are dendritic or other antigen presenting cells.
 32. The method ofclaim 23, wherein said subject suffers from a hyperproliferativedisease.
 33. The method of claim 1 further comprising additionallyadministering chemotherapy, immunotherapy, surgery, biotherapy,radiotherapy or a combination thereof.
 34. A method of treating ahyperproliferative disease comprising administering intratumorally to asubject a lactoferrin composition in combination with chemotherapy,biotherapy, immunotherapy, surgery or radiotherapy.